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Germline gain-of-function mutations in AFF4 cause a developmental syndrome functionally linking the super elongation complex and cohesin.

Izumi K, Nakato R, Zhang Z, Edmondson AC, Noon S, Dulik MC, Rajagopalan R, Venditti CP, Gripp K, Samanich J, Zackai EH, Deardorff MA, Clark D, Allen JL, Dorsett D, Misulovin Z, Komata M, Bando M, Kaur M, Katou Y, Shirahige K, Krantz ID - Nat. Genet. (2015)

Bottom Line: Transcriptional elongation is critical for gene expression regulation during embryogenesis.The super elongation complex (SEC) governs this process by mobilizing paused RNA polymerase II (RNAP2).Direct molecular interaction of the SEC, cohesin and RNAP2 was demonstrated.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. [2] Research Center for Epigenetic Disease, Institute for Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Transcriptional elongation is critical for gene expression regulation during embryogenesis. The super elongation complex (SEC) governs this process by mobilizing paused RNA polymerase II (RNAP2). Using exome sequencing, we discovered missense mutations in AFF4, a core component of the SEC, in three unrelated probands with a new syndrome that phenotypically overlaps Cornelia de Lange syndrome (CdLS) that we have named CHOPS syndrome (C for cognitive impairment and coarse facies, H for heart defects, O for obesity, P for pulmonary involvement and S for short stature and skeletal dysplasia). Transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analyses demonstrated similar alterations of genome-wide binding of AFF4, cohesin and RNAP2 in CdLS and CHOPS syndrome. Direct molecular interaction of the SEC, cohesin and RNAP2 was demonstrated. These data support a common molecular pathogenesis for CHOPS syndrome and CdLS caused by disturbance of transcriptional elongation due to alterations in genome-wide binding of AFF4 and cohesin.

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Disease mechanism of CHOPS syndrome. (a) Decreased proteosomal degradation of mutant AFF4 in 293T cells. Western blot demonstrates disappearance of WT AFF4 bands with the addition of SIAH1 vector. However, such disappearance was not observed in 293T cells overexpressing mutant AFF4 vectors. The numbers beneath AFF4 bands indicate the signal intensities normalized to the band intensity of AFF4 only transfection condition of each category and alpha tubulin. (b, c) Expression level of MYC/JUN gene in patient-derived skin fibroblasts and 293T cell line with AFF4 overexpression. GM01652, GM02036 and GM08398 are control fibroblast cell lines. Elevation of MYC and JUN expression were observed in CHOPS syndrome skin fibroblast (2b) and 293T AFF4 overexpression model (2c). MYC and JUN expression was normalized against TBP. Error bars demonstrate mean ± 2 standard deviations. **P<0.01, ***P<0.001, two-tailed t-test, n= 3 per group.
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Figure 2: Disease mechanism of CHOPS syndrome. (a) Decreased proteosomal degradation of mutant AFF4 in 293T cells. Western blot demonstrates disappearance of WT AFF4 bands with the addition of SIAH1 vector. However, such disappearance was not observed in 293T cells overexpressing mutant AFF4 vectors. The numbers beneath AFF4 bands indicate the signal intensities normalized to the band intensity of AFF4 only transfection condition of each category and alpha tubulin. (b, c) Expression level of MYC/JUN gene in patient-derived skin fibroblasts and 293T cell line with AFF4 overexpression. GM01652, GM02036 and GM08398 are control fibroblast cell lines. Elevation of MYC and JUN expression were observed in CHOPS syndrome skin fibroblast (2b) and 293T AFF4 overexpression model (2c). MYC and JUN expression was normalized against TBP. Error bars demonstrate mean ± 2 standard deviations. **P<0.01, ***P<0.001, two-tailed t-test, n= 3 per group.

Mentions: A missense mutation in the ALF homology domain of Aff1 (Af4) was reported in the robotic mouse, an ataxia mouse model created by ENU mutagenesis10. The pathogenetic mechanism of this missense mutation is a gain-of-function effect due to decreased clearance of the protein by SIAH1 ubiquitin E3 ligase11. Given the vicinity of the location of the missense mutations in our probands to that described in the robotic mouse, we hypothesized that the missense mutations found in the three probands would likewise disrupt the ubiquitinylation-dependent proteasomal degradation of the AFF4 protein. To test this hypothesis, we created an AFF4 and SIAH1 overexpression model using HEK293T cells. When a wild type (WT) AFF4 expression vector was transfected with an SIAH1 expression vector, the amount of AFF4 protein was significantly decreased; however the AFF4 constructs containing the missense mutations found in the three CHOPS syndrome probands hindered the degradation of AFF4 with SIAH1 overexpression (Fig. 2a). The addition of MG132, which is a proteosomal degradation inhibitor, resulted in the recovery of AFF4 bands.


Germline gain-of-function mutations in AFF4 cause a developmental syndrome functionally linking the super elongation complex and cohesin.

Izumi K, Nakato R, Zhang Z, Edmondson AC, Noon S, Dulik MC, Rajagopalan R, Venditti CP, Gripp K, Samanich J, Zackai EH, Deardorff MA, Clark D, Allen JL, Dorsett D, Misulovin Z, Komata M, Bando M, Kaur M, Katou Y, Shirahige K, Krantz ID - Nat. Genet. (2015)

Disease mechanism of CHOPS syndrome. (a) Decreased proteosomal degradation of mutant AFF4 in 293T cells. Western blot demonstrates disappearance of WT AFF4 bands with the addition of SIAH1 vector. However, such disappearance was not observed in 293T cells overexpressing mutant AFF4 vectors. The numbers beneath AFF4 bands indicate the signal intensities normalized to the band intensity of AFF4 only transfection condition of each category and alpha tubulin. (b, c) Expression level of MYC/JUN gene in patient-derived skin fibroblasts and 293T cell line with AFF4 overexpression. GM01652, GM02036 and GM08398 are control fibroblast cell lines. Elevation of MYC and JUN expression were observed in CHOPS syndrome skin fibroblast (2b) and 293T AFF4 overexpression model (2c). MYC and JUN expression was normalized against TBP. Error bars demonstrate mean ± 2 standard deviations. **P<0.01, ***P<0.001, two-tailed t-test, n= 3 per group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4380798&req=5

Figure 2: Disease mechanism of CHOPS syndrome. (a) Decreased proteosomal degradation of mutant AFF4 in 293T cells. Western blot demonstrates disappearance of WT AFF4 bands with the addition of SIAH1 vector. However, such disappearance was not observed in 293T cells overexpressing mutant AFF4 vectors. The numbers beneath AFF4 bands indicate the signal intensities normalized to the band intensity of AFF4 only transfection condition of each category and alpha tubulin. (b, c) Expression level of MYC/JUN gene in patient-derived skin fibroblasts and 293T cell line with AFF4 overexpression. GM01652, GM02036 and GM08398 are control fibroblast cell lines. Elevation of MYC and JUN expression were observed in CHOPS syndrome skin fibroblast (2b) and 293T AFF4 overexpression model (2c). MYC and JUN expression was normalized against TBP. Error bars demonstrate mean ± 2 standard deviations. **P<0.01, ***P<0.001, two-tailed t-test, n= 3 per group.
Mentions: A missense mutation in the ALF homology domain of Aff1 (Af4) was reported in the robotic mouse, an ataxia mouse model created by ENU mutagenesis10. The pathogenetic mechanism of this missense mutation is a gain-of-function effect due to decreased clearance of the protein by SIAH1 ubiquitin E3 ligase11. Given the vicinity of the location of the missense mutations in our probands to that described in the robotic mouse, we hypothesized that the missense mutations found in the three probands would likewise disrupt the ubiquitinylation-dependent proteasomal degradation of the AFF4 protein. To test this hypothesis, we created an AFF4 and SIAH1 overexpression model using HEK293T cells. When a wild type (WT) AFF4 expression vector was transfected with an SIAH1 expression vector, the amount of AFF4 protein was significantly decreased; however the AFF4 constructs containing the missense mutations found in the three CHOPS syndrome probands hindered the degradation of AFF4 with SIAH1 overexpression (Fig. 2a). The addition of MG132, which is a proteosomal degradation inhibitor, resulted in the recovery of AFF4 bands.

Bottom Line: Transcriptional elongation is critical for gene expression regulation during embryogenesis.The super elongation complex (SEC) governs this process by mobilizing paused RNA polymerase II (RNAP2).Direct molecular interaction of the SEC, cohesin and RNAP2 was demonstrated.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. [2] Research Center for Epigenetic Disease, Institute for Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Transcriptional elongation is critical for gene expression regulation during embryogenesis. The super elongation complex (SEC) governs this process by mobilizing paused RNA polymerase II (RNAP2). Using exome sequencing, we discovered missense mutations in AFF4, a core component of the SEC, in three unrelated probands with a new syndrome that phenotypically overlaps Cornelia de Lange syndrome (CdLS) that we have named CHOPS syndrome (C for cognitive impairment and coarse facies, H for heart defects, O for obesity, P for pulmonary involvement and S for short stature and skeletal dysplasia). Transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analyses demonstrated similar alterations of genome-wide binding of AFF4, cohesin and RNAP2 in CdLS and CHOPS syndrome. Direct molecular interaction of the SEC, cohesin and RNAP2 was demonstrated. These data support a common molecular pathogenesis for CHOPS syndrome and CdLS caused by disturbance of transcriptional elongation due to alterations in genome-wide binding of AFF4 and cohesin.

Show MeSH
Related in: MedlinePlus